Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of numerous cellular functions. Protein phosphorylation by protein kinase C (PKC) has been linked to the regulation of a plethora of cellular events. A physiological target of PKC, neurogranin (Ng), is also modified by nitric oxide and other oxidants. Ng binds calmodulin (CaM) with high affinity in the presence of low levels of calcium. Rat bovine, chicken, and human Ng have identical PKC phosphorylation site/CaM-binding domains. Phosphorylation by PKC reduces their binding affinity for CaM and thus favors activation of CaM-dependent enzymes. Rat brain Mg contains four cysteines located at positions 3,4,9, and 51. Site-directed mutagenesis showed Cys9 and Cys51 to be essential for the oxidant-mediated intramolecular disulfide bridging that attenuates the CaM-binding affinity of Ng. Bovine Ng and a human variant (Ng-2) lack Cy351, and chicken Ng and another human variant (Ng-1), lack both Cys9 and Cys51. Based on these structural variations we predict that the oxidant- mediated regulation of Ng function, unlike that by PKC, is variable among species. A novel 28 kDa PKC/CK2 substrate, termed HASPP28, has been identified and its cDNA cloned. This protein is a substrate of CK2 in NIE115 neuroblastoma cells, and its phosphorylation and protein level are cell cycle-regulated. Genomic clones containing more than 90 percent of the coding region and 1.2 kb upstream of the transcription start site, without intron sequences, have been characterized. Promoter activity was defined with nested deletion constructs fused to a luciferase reporter gene; RGF, dBu-cAMP, and PMA caused 2-3 fold stimulation whereas PDGF, bFGF, and insulin had no effect. Immunoprecipitated HASPP28 contains a CK2-like kinase that phosphorylates endogenous HASPP28 or exogenous recombinant protein. The HASPP28-associated protein is being investigated by yeast two-hybrid system. The functional roles of the various PKC isozymes in the control of cellular differentiation were analyzed in NIE115 neuroblastoma cells. PKC-activating phorbol ester counteracts the cAMP- induced neurite outgrowth (NOG) of these cells by selective translocation and downregulation of PKC alpha or delta, with reduced levels of the respective PKCs, were more sensitive to dBu-alphaAMP for NOG. These two antisense PKC stable transfectants were also less responsive to PMA in counteracting cAMP-induced NOG, suggesting that PKC alpha and delta are involved in regulating NOG.